JP7221545B2 - Phosphatidylserine targeting fusion molecules and methods of their use - Google Patents

Description

This patent application claims the benefit of priority from US Provisional Application No. 62/536,107, filed July 24, 2017, the contents of which are hereby incorporated by reference in their entirety.

The present invention provides fusion molecules comprising a cytokine and a polypeptide that targets the fusion protein to phosphatidylserine (PS), pharmaceutical compositions comprising these fusion molecules, and targeting cytokines to lesion sites. , and methods of using these fusion molecules in treating diseases or conditions that are responsive to cytokine treatment.

BACKGROUND Phosphatidylserine (PS) is an anionic phospholipid externalized on the surface of apoptotic cells, apoptotic blebs, exosomes, stressed tumor cells and tumor vasculature, but immune in the tumor microenvironment. It is an inhibitory molecule (Birge et al. Cell Death and Differentiation 2016 23:962-978). Upregulation of PS in the tumor microenvironment due to hypoxia and other metabolic stresses, high apoptotic indices of apoptotic cells, and the release of tumor-derived exosomes has been observed in virtually all solid tumors (He et al. al. Clin. Cancer Res. 2009 15: 6871-6880). Upregulated PS then interacts with overexpressed Tyro3, Axl and Mer (TAM) receptors on tumor cells and on infiltrating bone marrow-derived phagocytes. Engagement of PS/PS receptors collectively causes PS-dependent efferocytosis and production of immunosuppressive cytokines such as IL-10 and TGF-β. (Huynh et al. J. Clin. Invest. 2002 109:41-50; Rothlin et al. Cell 2007 131: 1124-1136).

In addition to the tumor microenvironment, PS is also expressed by infected cells, especially virus-infected cells (Soares et al. Nat. Med. 2008 207:763-776; Dowall et al. J. Immunol. Res. 2015 347903). In addition, enveloped viruses of various species expose PS on their surface and use it to suppress immune responses and promote tolerance to viral antigens, as well as TAMs as a mechanism for virus entry into cells. Utilizes receptors (Birge et al. Cell Death. Differ. 2016 23:962-978). Viral particles opsonized with growth arrest-specific gene 6 (GAS6) and protein S (Pros1) can interact with TAMs, be efferocytosed, uncoated in endosomes, and enter the cytoplasm. It is shown.

However, although PS is constitutively elevated in the tumor microenvironment and on the surface of enveloped viruses, under normal physiological conditions, even in tissues with high cell turnover rates, such as thymus and spleen, Few un-cleared apoptotic cells are observed. Therefore, PS is not detected in normal tissues (Gerber et al. Clin. Cancer Res. 2011 17:6888-6896).

PS-targeting has therefore been disclosed as a possible means for the local delivery of therapeutic agents to sites with lesions in which PS is upregulated as part of the stress response.

U.S. Pat. No. 6,211,142 is a composition comprising a functionally active gas6 variant having less gamma-carboxylation than gas6 from an endogenous source, as well as products comprising the same. disclose said products for activation of Rse receptor protein tyrosine kinase and promotion of proliferation, survival and/or differentiation of Rse receptor containing cells such as neuronal and glial cells.

Canadian Patent Invention No. 2909669 describes therapeutic compositions that inhibit AXL, MER or Tyro3 protein activity, e.g. by inhibiting the binding interaction of AXL, MER or Tyro3 with its ligand GAS6. Compositions and methods are disclosed for treating viral infections in mammals by administering doses. Also, by administering one or more inhibitors of AXL, MER and/or Tyro3 activity, inhibitors of GAS6 activity, or inhibitors of AXL, MER or Tyro3-GAS6 interactions, phosphatidylserine in mammalian patients Also disclosed are methods of treating, reducing or inhibiting phosphatidylserine harboring viral infections.

Japanese Patent No. 5478285 discloses targeting tumor vasculature using conjugates that bind to phosphatidylserine. Disclosed targeting agents include killing tumor vascular endothelial cells, causing coagulation in tumor vasculature; or disrupting tumor vasculature, causing tumor necrosis and/or tumor regression; Included are anti-phosphatidylserine antibodies or antigen-binding fragments thereof, annexins, or phosphatidylserine-binding fragments.

Japanese Patent No. 4743672 also describes a cancer treatment that kills tumor vascular endothelial cells, causes coagulation in tumor vasculature, or causes tumor necrosis and/or tumor regression by destroying tumor vasculature. disclose anti-phosphatidylserine antibodies.

US Pat. No. 6,312,694 discloses aminophospholipid-targeted diagnostic and therapeutic antibody-therapeutic agent constructs for use in tumor intervention.

Kimani et al. (Scientific Reports 2017 7:43908) disclose small molecule inhibitors that target the extracellular domain of Axl at the interface between the Ig-1 ectodomain of Axl and the Lg-1 of Gas6. , which effectively blocks Gas6-induced Axl receptor activation and suppresses H1299 lung cancer tumor growth in a mouse xenograft NOD-SCIDγ model.

Preclinical studies have also been performed in the population with PS-targeting antibodies that bind PS with high affinity, either directly or conjugated to the serum protein β2-glycoprotein 1 (DeRose et al. Immunotherapy 2011 3:933-944: Huang et al. Cancer Res. 2005 65:4408-4416). These antibodies target endothelial cells in the tumor microenvironment (Ran et al. Cancer Res. 2002 62:6132-6140) and exhibit anti-tumor activity (de Freitas Balanco et al. Curr. Biol. 2001 11:1870-1873) and to enhance the activity of standard treatments in multiple preclinical tumor models (Beck et al. Int. J. Cancer 2005 118:2639-2643; He et al. Clin. Cancer Res 2009 15:6871-6880), was shown.

In addition, the PS-targeting antibody bavituximab has been evaluated in multiple clinical trials (Chalassani et al. Cancer Med. 2015 4:1051-1059; Digumarti et al. Lung Cancer 2014 86:231-236; Gerber et al. Clin. Cancer Res. 2011 17:6888-6896). However, despite the hype surrounding the promise of a PS-targeting monoclonal antibody (mAb), Peregrine Pharmaceuticals' latest phase III SUNRISE clinical trial had disappointing results and no new patient recruitment in 2016. As a result, it was discontinued. Further studies are beginning to evaluate the therapeutic efficacy of this antibody in combination with an anti-PD-L1 antibody for the treatment of solid tumors (world wide web globenewswire.com/news-release/2017/06/05/). 1008110/0/en/Peregrine-Pharmaceuticals-Presents-Preliminary-Correlative-Analysis-of-PD-L1-Expression-from-SUNRISE-Trial-at-ASCO-2017.html, June 5, 2017).
There is a need to develop more effective PS-targeted derivatives as second- or next-generation immunobiologicals.

SUMMARY Aspects of the present invention relate to fusion molecules that include a cytokine and a polypeptide that targets the fusion molecule to phosphatidylserine (PS).
In one non-limiting embodiment, the polypeptide of the fusion molecule comprises PS-binding ligands of Tyro3, Axl and Mer receptors, also referred to herein as TAM ligands.

In one non-limiting embodiment, the polypeptide of the fusion molecule comprises a PS-linked domain of growth arrest-specific gene 6 (GAS6) or protein S (Prosl). .
In one non-limiting embodiment, the cytokine of the fusion molecule is an immunostimulatory cytokine.
In another non-limiting embodiment, the cytokine of the fusion molecule is an immunosuppressive cytokine.

Another aspect of the invention relates to pharmaceutical compositions comprising the fusion molecules of the invention.
Another aspect of the invention relates to methods of targeting cytokines to lesion sites in a subject by administering a fusion molecule of the invention or a pharmaceutical composition comprising a fusion molecule of the invention.

Another aspect of the invention is immunosuppression resulting from PS recognition by endogenous PS ligands and receptors at a lesion site in a subject by administering a fusion molecule of the invention or a pharmaceutical composition comprising a fusion molecule of the invention. It relates to a method of inhibiting

Another aspect of the invention is to activate one or more cytokine-specific biological activities at the lesion site by administering a fusion molecule of the invention or a pharmaceutical composition comprising a fusion molecule of the invention. Regarding the method.
Another aspect of the invention relates to methods of minimizing the systemic effects of cytokines by administering cytokines via a fusion molecule of the invention or a pharmaceutical composition comprising a fusion molecule of the invention.

Yet another aspect of the invention relates to methods of treating a disease, disorder or condition responsive to cytokine treatment by administering a fusion molecule of the invention or a pharmaceutical composition comprising a fusion molecule of the invention. .
In one non-limiting embodiment, the disease, disorder or condition treated by the present invention is cancer, infection or an inflammatory condition or disorder.

FIG. 1 provides schematic representations of various non-limiting embodiments of the fusion molecules of the invention. In particular, schematic representations of recombinant IFN fusion molecules and recombinant Gas6-IFN fusion molecules containing the PS-binding Gla domain and the EGF repeats of Gas6 are provided. Gas6-IFN fusion molecules are designed to redirect immunosuppressive signals into immunogenic signals that activate host anti-tumor immunity. Linker sequences and variants are defined in this application.

FIG. 2 depicts a model of the type III IFN (IFN-λ) and type I IFN (IFN-α/β) receptor system. IFN-λ and type I IFNs use different heterodimeric receptor complexes. IFN-λ engages native IFN-λR1 and IL-10R2, whereas IFN-αR1 and IFN-αR2 form the active type I IFN receptor complex. Engagement of IFN-α or IFN-λ receptors results in phosphorylation of the receptor-associated JAK kinases JAK1 and Tyk2, followed by phosphorylation of STAT1 and STAT2, which interact with the DNA-binding protein IRF9, resulting in the release of IFNs. It leads to the formation of a transcriptional complex called stimulated gene factor 3 (ISGF3), which binds to IFN-stimulated response elements (ISREs) to control transcription of IFN-stimulated genes (ISGs).

FIG. 3 provides a schematic representation of the proposed immunogenic function of Gas6-IFN-β and/or IFN-λ2 fusion molecules of the invention in PS-enriched tumor microenvironments or sites of viral infection. Gas6 (via its Gla and EGF-like domains) has been proposed to act as a PS sensor and respond to the extent of PS expressed in the tissue microenvironment. Gas6 will respond to the concentration of expressed PS and localize cytokines to tissues in a PS-dependent manner. At lower expressed PS concentrations, IFN activity is expected to be low (native cytokine activity), whereas at higher concentrations (either in the tumor microenvironment or in virus-infected cells/tissues), IFN The activity is expected to be amplified, increasing cytokine activity and leading to enhanced anti-tumor immunity and anti-viral responses. Legends in the figure identify potential target cell types, as well as expected phenotypic consequences. For example, in tumor cells, targeting Gas6-IFN results in increased expression of MHC class I antigens and co-stimulatory molecules, which leads to increased expression/presentation of tumor antigens, anti-angiogenic chemokines. It is predicted to lead to increased production and increased infiltration of immune cells. In antigen-presenting cells, Gas6-IFN fusion molecules are predicted to increase the expression of MHC class I and MHC class II antigens as well as increase maturation of dendritic cells and increase cross-presentation of tumor antigens. be.

Figure 4 depicts the principle of a "second generation" PS-targeted biologic. To date, PS-targeted mAbs have been developed that bind to expressed PS and essentially mask it. The Gas6-IFN fusion molecules developed herein are designed to convert tolerogenic signals into immunogenic signals by targeting IFN to PS-rich TMEs. . In addition, Gas6-targeted IFNs induce PDL1 and are therefore very well adapted for use as combination therapeutics with anti-PD1/anti-PDL1. Gas6-IFN biologic properties are provided in the description.

Figures 5(A) and (B) show the production, expression, and detection of His-tagged Gas-IFN proteins. The Gas6-IFN fusion molecule was cloned and expressed in HEK293, E0771 and Expi293 cells (for larger scale production). Secretion of the recombinant fusion molecule into the cell supernatant was analyzed by immunoblotting with an anti-His mAb in HEK293T cell supernatants harvested 48 hours after transfection, demonstrating the presence of a His-tagged protein at the expected molecular weight (Fig. Upper panel; (A)). Immunoblots with anti-Gla mAb (bottom panel; (B)), probed with a γ-carboxylation specific antibody, show γ-carboxylation. As noted, with an anti-Gla-specific mAb, all Gas6 fusion molecules (last 4 lanes) are γ-carboxylated (PS binding) when cells are grown in the presence of vitamin K. requirements). These results demonstrate that the protein is active as a PS binding protein, which is a requirement of the claims in this application.

FIG. 6. Lysates of IFN-λR-γR1 reporter cell lines treated with HEK293T cell supernatants containing recombinant IFN-λ2 or Gas6(Gla+EGF)-IFN-λ2 fusion molecules with or without apoptotic cells for 30 minutes. shows the activity of the Gas6-IFN-λ2 fusion molecules of the invention as measured by detecting the extent of Stat1 activation (Tyr phosphorylation of Stat1, pStat1) by immunoblot of . Immunoblots for pStat1 showed a PS-binding-dependent increase in IFN-λ receptor activation by the fusion molecule, especially at high concentrations of PS (1:1000-reporter cells/apoptotic cells (AC); lane 7 and 10 comparison).

FIG. 7 provides a schematic of a Gas6(Gla+EGF)-IFN-β-IFN-λ2 fusion molecule containing a phosphotag and a CLIP tag to label the peptide for protein purification and detection. In addition to the His-tagged proteins, phosphorylation tags (for ³² P labeling) and CLIP tags (for fluorescently labeled proteins) were engineered into fusion molecules containing type I and type III IFNs. These latter tags were introduced for in vivo labeling, utility, and localization. The left side of the figure reiterates the domain structure of Gas6, including the Gla-binding region to PS, EGF repeats, and the LG domain that binds the TAM receptor but is displaced by cytokine(s). .

FIG. 8 shows that in contrast to IFN-β-IFN-λ2 or Gas6-IFN-β-IFN-λ2 proteins prepared in the presence of warfarin, Gas6-IFN-β-IFN-λ2 protein was PS-positive. It is shown to bind to and thereby precipitate apoptotic cells. Gas6-IFN-β-IFN-λ2 protein was produced in HEK293 cells in the presence of vitamin K (vit K), which is required for γ-carboxylation, and warfarin (War), which inhibits γ-carboxylation. Proteins were then incubated with apoptotic cells (AC) followed by sedimentation of apoptotic cells by centrifugation. The presence of IFN activity co-precipitated with apoptotic cells detects the extent of Stat1 activation (pStat1) by immunoblotting lysates of the IFN-λR-γR1 reporter cell line similar to the experiment described in FIG. It was measured by Comparing lanes 4, 7, and 10, only the γ-carboxylated Gas6-IFN-β-IFN-λ2 fusion molecule prepared with vitamin K is active when co-precipitated with PS-positive apoptotic cells.

FIG. 9 shows partial purification and detection of His-tagged IFN-β-IFN-λ2 and His-tagged Gas6-IFN-β-IFN-λ2. Left panel shows immunoblotting with anti-His mAb, right panel shows Coomassie blue staining and level of protein purity.

Figure 10 shows the antiviral activity of Gas6-IFN-β-IFN-λ2 fusion molecules of the invention. Mouse intestinal epithelial cells (mIEC) were pretreated with HEK293T cell supernatants containing IFN-β-IFN-λ2 or Gas6-IFN-β-IFN-λ2 fusion molecules. After 24 hours of pretreatment, cells were treated with vesicular stomatitis virus (VSV) for 24 hours and antiviral activity of fusion molecules was analyzed. Cell viability was measured using the MTT assay.

FIG. 11 shows the relative antiviral efficacy of IFN-β-IFN-λ2 or Gas6-IFN-β-IFN-λ2 fusion molecules. The results of the antiviral assay, shown in FIG. 10, were normalized per amount of IFN-β-IFN-λ2 and Gas6-IFN-β-IFN-λ2 proteins in HEK293 supernatants to determine their relative antiviral potency. (activity/mg) was determined. Both immunoblotting and Coomassie staining (Fig. 9) showed that in supernatants of HEK293 cells used to generate samples for the antiviral assay shown in Fig. 10, the amount of Gas6-IFN fusion molecules It shows less than the amount of molecules (5-fold). In these assays, fusion molecule-containing supernatants were used starting from the same dilution factor, so when normalized with a lower concentration of Gas6-IFN-β-IFN-λ2 fusion molecule, Gas6- The IFN-β-IFN-λ2 fusion molecule is more active (5-fold) than the IFN-β-IFN-λ2 fusion molecule.

Figure 12 shows that Gas6-IFN-β-IFN-λ2 protein retains the ability to induce expression of MHC class I antigens in mouse intestinal epithelial cells, supporting its immunogenic activity. Mouse intestinal epithelial cells were treated for 72 hours with cell culture supernatants containing IFN-β-IFN-λ2 or Gas6-IFN-β-IFN-λ2 fusion molecules, and the expression of MHC class I proteins and MHC-specific antibodies increased. Analyzed by flow cytometry using.

Figure 13 shows that the Gas6-IFN-β-IFN-λ2 protein retains the ability to induce PD-L1 expression in mouse intestinal epithelial cells. Mouse intestinal epithelial cells were treated for 72 hours with cell culture supernatants containing IFN-β-IFN-λ2 or Gas6-IFN-β-IFN-λ2 fusion molecules, and the expression of MHC class I proteins was measured by PD-L1-specific antibodies. was analyzed by flow cytometry using .

Figure 14 shows that the Gas6-IFN-β-IFN-λ2 fusion molecule has superior anti-tumor activity as a single entity molecule than when co-expressed as separate entities. EO771 cells, a mouse breast cancer cell line, were stably transfected with an empty vector or an expression vector encoding either Gas6-IFN-β, Gas6-IFN-λ2 or Gas6-IFN-β-IFN-λ2 fusion molecules. were transected and implanted into the mammary fat pad of syngeneic C57BL/6 mice. to a 50:50 mixture of each protein of 0.5×10 ⁵ E0771 cells constitutively secreting Gas6-IFN-β and 0.5× ^{10 5} E0771 cells constitutively secreting Gas6-IFN-λ2 , tumor growth (tumor volume) measurements after injection of 10 ⁵ mock (empty vector) EO771 cells or 10 ⁵ E0771 cells constitutively secreting the Gas6-IFN-β-IFN-λ2 fusion molecule. shows the results of TF indicates tumor-free mice (tumor-free mice).

FIG. 15 shows the tumor volume of each animal on day 29 for the experiment outlined in FIG.

Figure 16 shows similar anti-tumor activity of IFN-β-IFN-λ2 and Gas6-IFN-β-IFN-λ2 fusion molecules. Similar to the experiments outlined in FIG. 14, 10 ⁵ EO771 cells mock (empty vector) or E0771 constitutively secreting IFN-β-IFN-λ2 or Gas6-IFN-β-IFN-λ2 fusion molecules. Results of tumor growth (tumor volume) measurements after injection of 10 ⁵ cells are shown. TF indicates tumor-free mice (tumor-free mice).

FIG. 17 shows the tumor volume of each animal on day 29 for the experiment outlined in FIG.

Figure 18 summarizes the advantages of the PS-targeted fusion molecules of the invention.

Figures 19A-19C show IFN-λ2 reporter activity of Gas6-IFN-λ2 fusion molecules of the invention. FIG. 19A shows Gas6(Gla)-IFN-λ2 and Gas6(Gla+EGF)-IFN-λ2 fusion molecules secreted into HEK293T cell supernatants harvested 48 h after transfection at predicted molecular weights of 37 and 70 kd. Immunoblot showing γ-carboxylation probed with a γ-carboxylation-specific antibody. Figures 19B and 19C show that the IFN-λR-γR1 reporter cell lines were treated with or without recombinant IFN-λ2 or Gas6(Gla)-IFN-λ2 (Fig. 19B) and Gas6(Gla+EGF)-IFN-λ2 ( FIG. 19C) shows the results of treatment with HEK293T cell supernatant containing fusion molecules for 30 minutes. pStat1 immunoblot shows a phosphatidylserine binding-dependent increase in activation of IFN-λ receptor cell lines by the fusion molecule.

Figures 20A-20C show IFN-λ2 functional activity of Gas6-IFN-λ2 fusion molecules of the invention. In FIG. 20A, human retinal pigment epithelial cells ARPE19 were pretreated with HEK293T cell supernatants containing recombinant IFN-λ2 or Gas6(Gla)-IFN-λ2 and Gas6(Gla+EGF)-IFN-λ2 fusion molecules. . Twelve hours after pretreatment, cells were treated with vesicular stomatitis virus (VSV) for 24 hours and the antiviral activity of fusion molecules was analyzed. Cell viability was measured using the MTT assay. FIG. 20A shows that the antiviral activity of the fusion molecule is equivalent to recombinant IFN-λ2. Figures 20B and 20C show the immunogenic protein calreticulin (Figure 20B) and MHC class as determined by flow cytometry of ARPE19 cells after 72 hours of treatment with recombinant IFN-λ2 or fusion molecules of the invention. Expression of I protein (Fig. 20C) is shown.

Figures 21A and 21B show the anti-tumor activity of Gas6-IFN-λ2 fusion molecules of the invention. FIG. 21A. Gas6(Gla)-IFN secreted from EO771, a murine breast cancer cell line stably expressing the fusion molecule, after treatment with vitamin K (Vit. K) or the gamma-carboxylation inhibitor warfarin (Warf). Immunoblot showing γ-carboxylation of -λ2 and Gas6(Gla+EGF)-IFN-λ2 fusion molecules. FIG. 21B shows tumor growth after injection of 0.1×10 ⁶ EO771 mock-transfected (empty vector) cells or Gas6(Gla+EGF)-IFN-λ2 fusion molecule-secreting cells into the mammary fat pad of C57BL/6 mice. The results of volumetric measurements are shown.

DETAILED DESCRIPTION While the immune system has the potential to eliminate pathogenic cells such as tumor cells, viruses and inflammatory cells involved in inflammatory diseases, a major barrier to effective immunotherapy is clinical It is the ability to elicit meaningful responses. To do so, the host must be able to overcome endogenous inhibitory mechanisms that limit the development of an effective immune response.

Cytokines are potent regulators of various immune functions and can be used to treat a wide range of pathological conditions, including cancer, infections, and immune and inflammatory diseases. Due to the undesirable side effects associated with systemic administration of many cytokines, it is highly desirable to target the cytokines to the lesion-bearing site in order to achieve local action of the cytokines.

Phosphatidylserine (PS) expression is a hallmark of cancer cells themselves, and dysregulated PS expression in the tumor microenvironment (TME) has been observed in a wide range of human cancers, including all solid tumors. It is a feature of Dysregulated PS in TME can occur in a variety of cell types, including apoptotic tumor cells, stressed tumor cells due to hypoxia and nutrient deprivation and various tumor-infiltrating cells, and stressed vascular endothelial cells at tumor sites.

In addition, cells undergoing stress from hypoxia and nutritional deprivation due to infection and/or inflammatory conditions also express PS. In addition, enveloped viruses expose PS on their surface.

Therefore, targeting cytokines to PS-rich regions serves as a means of delivering cytokines to sites of tumor, viral infection, and inflammation, while minimizing their systemic effects.

PS concentrations on the cell surface appear to reflect cellular stress levels; changes in PS concentrations are associated with TAMs (Tyro3, Axl and Mer) activated by the PS-binding TAM ligands Gas6 and Pros1. It is sensed by a group of collectively known receptors. These ligands act as bridging molecules, interacting with PS through their N-terminal Gla domains, and bind and activate TAMs through their C-terminal LG domains. TAM activation is strictly PS dependent, with PS concentration acting as a rheostat of TAM activation strength.

The present invention provides engineered bifunctional PS-targeting cytokine fusion immunobiologicals and their use in targeting cytokines to tumor sites, sites of viral infection, and sites of inflammation. Provide usage instructions. Unlike PS-targeted mAbs, which bind to PS and passively block PS interactions with cognate receptors on tumor and myeloid cells, the fusion molecules of the present invention are capable of suppressing immune stimulation in a PS-rich microenvironment. It is designed so that the intensity of sexual cytokine signaling can be matched to PS concentration. Thus, in the presence of PS, these PS-targeted cytokine fusion molecules induced stronger and sustained cytokine receptor activation, leading to increased biology of PS-targeted cytokine fusion molecules compared to unmodified cytokines. bring about active activity.

Furthermore, activation of PS receptors, which can be caused by direct binding to PS or PS-interacting ligands, leads to an immunosuppressive state that is normally established and maintained, eg, during tumor progression.

The PS-targeted cytokine fusion molecules of the present invention block their ability by providing immune activation through cytokine-specific activity and by competing for PS binding with endogenous PS ligands and receptors. are expected to induce an immunosuppressive state and redirect back to an immunosuppressive state.

Thus, the bifunctional PS-targeting cytokine fusion molecules of the invention are expected to bind PS in stress cells and localize immunostimulatory cytokine signaling to areas of high expressed PS density. In doing so, the fusion molecules of the invention redirect tolerogenic signals generated by sustained engagement of immunosuppressive PS receptors to immunogenic signals derived from the PS->cytokine receptor axis. expected to let

Therefore, the invention provides a fusion molecule comprising a cytokine or portion thereof and a polypeptide that targets the fusion protein to PS. The fusion molecules of the invention that have been developed demonstrate that they provide PS-targeted and localized delivery of cytokines; that they block PS recognition by endogenous PS ligands and receptors; It has three unique features of actively changing the immune activation balance from PS-induced immunosuppression to immune activation that matches the PS level by activating specific biological activities. ing. Therefore, according to the present invention, pharmaceutical compositions comprising these fusion molecules, as well as targeting cytokines to lesion sites in subjects, inhibiting immunosuppression resulting from PS recognition by endogenous PS ligands in lesion sites in subjects, Activating one or more cytokine-specific biological activities at the lesion site, minimizing the systemic effects of cytokines, and/or treating diseases, disorders or conditions responsive to cytokine treatment Also provided are methods of using the fusion molecules and pharmaceutical compositions in . In one non-limiting embodiment, the disease, disorder or condition targeted and/or treated by the present invention is cancer, infection or an inflammatory condition or disorder.

For the purposes of the present invention, the terms "fusion protein" and "fusion molecule" are used interchangeably and encompass polypeptides, proteins and/or molecules made from portions derived from different sources. means Such fusion molecules are made by joining two or more genes or fragments thereof that originally encoded separate proteins or portions thereof. Translation of these fusion genes or portions thereof results in single or multiple polypeptides with functional properties derived from each original protein. In one non-limiting embodiment, the fusion molecule or protein is engineered by recombinant DNA technology for use in biological research or therapy.

For the purposes of the present invention, by "a portion thereof" is a fragment that is shorter in length than the full-length cytokine protein and retains at least a portion of its functional activity relative to the full-length protein and/or receptor It is meant to remain bound to at least one of the subunits.

Various immunostimulatory or immunosuppressive cytokines or portions thereof known to those skilled in the art can be included in the fusion molecules of the invention. In one non-limiting embodiment, the selected cytokine has desired activity at pathogenic PS-rich sites. In one non-limiting embodiment, the cytokine is an interferon (IFN) or portion thereof. IFNs are pluripotent cytokines and play an important role in establishing pleiotropic antiviral and antitumor responses. Examples of cytokines that can be included in the fusion molecules of the invention include interferon-α (IFN-α), interferon-β (IFN-β), interferon-λ1 (IFN-λ1), interferon-λ2 (IFN-λ2). , interferon-λ3 (IFN-λ3), interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL -15), interleukin 22 (IL-22), interleukin 33 (IL-33), amphiregulin (AREG), combinations or portions thereof, but are in no way limited thereto. Some cytokines, such as IFN-β, IFN-λ1, IFN-λ-2 and IFN-λ3, have unpaired Cys residues that can be replaced to improve folding and purification of fusion molecules. have Cytokine variants with lower affinity for their corresponding receptors can also be used to generate fusion PS-targeted cytokine proteins to reduce their ability to signal through their receptor complexes. , when activated by fusion PS-targeted cytokines, allow their increased activation in the presence of PS via PS-mediated oligomerization of cytokine receptor complexes.

Fusion molecules of the invention further include polypeptides that target the fusion molecule to PS. These fusion molecules can include various polypeptides that target the fusion molecule to PS. Examples of PS-targeting polypeptides that may be included in the fusion molecules of the invention include cerebral angiogenesis inhibitor 1 (BAI1), annexins, particularly annexins A5 and B12, T cell immunoglobulins and mucin receptors 1, 3 and 4. (TIM-1, TIM-3 and TIM-4), stabilins 1 and 2, and milk fat globule-EGF factor 8 protein (MFGE8). In one non-limiting embodiment, the fusion molecule comprises a polypeptide comprising PS-binding ligands of Tyro3, Axl and/or Mer (TAM) receptors. In one non-limiting embodiment, the fusion molecule comprises a polypeptide comprising the PS-linked domain of growth arrest specific gene 6 (GAS6) or protein S (Prosl). In one non-limiting embodiment, the fusion molecule comprises a polypeptide comprising the N-terminal Gla domain of Gas6 or Pros1. Non-limiting examples of polypeptides useful in fusion molecules of the invention include:

Mouse Gas6 Gla domain with signal peptide and pro-domain:
MPPPPGPAAALGTALLLLLLASESSHTVLLRAREAAQFLRPRQRRAYQVFEEAKQGHLERECVEEVCSKEEAREVFENDPETEYFYPRYQE (SEQ ID NO: 1);

Mouse Gas6 Gla domain with pro-domain and no signal peptide:
TVLLRAREAAQFLRPRQRRAYQVFEEAKQGHLERECVEEVCSKEEAREVFENDPETEYFYPRYQE (SEQ ID NO: 2);

Gla domain of mouse Gas6 without signal peptide and pro-domain:
AYQVFEEAKQGHLERECVEEVCSKEEAREVFENDPETEYFYPRYQE (SEQ ID NO: 3);

Gla domain of human Gas6 with signal peptide and pro-domain:
MAPSLSPGPAALRRAPQLLLLLLAAECALAALLPAREATQFLRPRQRRAFQVFEEAKQGHLERECVEELCSREEAREVFENDPETDYFYPRYLD (SEQ ID NO: 4);

Gla domain of human Gas6 with pro-domain and without signal peptide:
ALLPAREATQFLRPRQRRAFQVFEEAKQGHLERECVEELCSREEAREVFENDPETDYFYPRYLD (SEQ ID NO: 5);

Gla domain of human Gas6 without signal peptide and pro-domain:
AFQVFEEAKQGHLERECVEELCSREEAREVFENDPETDYFYPRYLD (SEQ ID NO: 6);

Gla domain of human Pros1 with signal peptide and pro-domain:
MRVLGGRCGALLACLLLVLPVSEANFLSKQQASQVLVRKRRANSLLEETKQGNLERECIEELCNKEEAREVFENDPETDYFYPKYLV (SEQ ID NO: 7);

Gla domain of human Pros1 with pro-domain and without signal peptide:
NFLSKQQASQVLVRKRRANSLLEETKQGNLERECIEELCNKEEAREVFENDPETDYFYPKYLV (SEQ ID NO: 8); and

Gla domain of human Pros1 without signal peptide and pro-domain:
ANSLLEETKQGNLERECIEELCNKEEAREVFENDPETDYFYPKYLV (SEQ ID NO: 9)
is included.

Additionally, in some embodiments, a polypeptide targeting a fusion molecule to PS may further comprise a domain that promotes oligomerization of the PS binding domain upon binding PS. A non-limiting example of a domain that promotes oligomerization of the PS-binding domain upon binding PS that may be included in the fusion molecules of the invention is the epidermal growth factor (EGF)-like domain of Gas6 or Pros1. Non-limiting examples include:

EGF-like domain of human GAS6
CINKYGSPYTKNSGFATCVQNLPDQCTPNPCDRKGTQACQDLMGNFFCLCKAGWGGRLCDKDVNECSQENGGCLQICHNKPGSFHCSCHSGFELSSDGRTCQDIDECADSEACGEARCKNLPGSYSCLCDEGFAYSSQEKACRDDECLQGRCEQVCVNSPGSYTCHCDGRGGLKLSQDMDTCE (SEQ ID NO: 10);

EGF-like domain of human Pros1
CLRSFQTGLFTAARQSTNAYPDLRSCVNAIPDQCSPLPCNEDGYMSCKDGKASFTCTCKPGWQGEKCEFDINECKDPSNINGGCSQICDNTPGSYHCSCKNGFVMLSNKKDCKDVDECSLKPSICGTAVCKNIPGDFECECPEGYRYNLKSKSCEDIDECSENMCAQLCVNYPGGYTCYCDGKKGFKLAQDQKSCE (SEQ ID NO: 11)
is included.

Thus, in one non-limiting embodiment, a fusion molecule of the invention may comprise a polypeptide comprising the N-terminal PS-linked domain and EGF-like oligomerization domain of GAS6 or Pros1. Non-limiting examples of such fusion molecules include:

Gla and EGF-like domains of human Gas6 with signal peptide and pro-domain:
MAPSLSPGPAALRRAPQLLLLLLAAECALAALLPAREATQFLRPRQRRAFQVFEEAKQGHLERECVEELCSREEAREVFENDPETDYFYPRYLDCINKYGSPYTKNSGFATCVQNLPDQCTPNPCDRKGTQACQDLMGNFFCLCKAGWGGRLCDKDVNECSQENGGCLQICHNKPGSFHCSCHSGFELSSDGRTCQDIDECADSEACGEARCKNLPGSYSCLCDEGFAYSSQEKACRDVDECLQGRCEQVCVNSPGSYTCHCDGRGGLKLSQDMDTCE（配列番号１２）；

Gla and EGF-like domains of human Gas6 with pro-domain and no signal peptide:
ALLPAREATQFLRPRQRRAFQVFEEAKQGHLERECVEELCSREEAREVFENDPETDYFYPRYLDCINKYGSPYTKNSGFATCVQNLPDQCTPNPCDRKGTQACQDLMGNFFCLCKAGWGGRLCDKDVNECSQENGGCLQICHNKPGSFHCSCHSGFELSSDGRTCQDIDECADSEACGEARCKNLPGSYSCLCDEGFAYSSQEKACRDVDECLQVCVNSPGSYTCHCDGGDT3 array number 1;

Gla and EGF-like domains of human Gas6 without signal peptide and pro-domain:
(SEQ ID NO: 1):

Gla and EGF-like domains of human Pros1 with signal peptide and pro-domain:
MRVLGGRCGALLACLLLVLPVSEANFLSKQQASQVLVRKRRANSLLEETKQGNLERECIEELCNKEEAREVFENDPETDYFYPKYLVCLRSFQTGLFTAARQSTNAYPDLRSCVNAIPDQCSPLPCNEDGYMSCKDGKASFTCTCKPGWQGEKCEFDINECKDPSNINGGCSQICDNTPGSYHCSCKNGFVMLSNKKDCKDVDECSLKPSICGTAVCKNIPGDFECECPEGYRYNLKSKSCEDIDECSENMCAQLCVNYPGGYTCYCDGKKGFKLAQDQKSCE（配列番号１５）：

Gla and EGF-like domains of human Pros1 with pro-domain and no signal peptide:
NFLSKQQASQVLVRKRRANSLLEETKQGNLERECIEELCNKEEAREVFENDPETDYFYPKYLVCLRSFQTGLFTAARQSTNAYPDLRSCVNAIPDQCSPLPCNEDGYMSCKDGKASFTCTCKPGWQGEKCEFDINECKDPSNINGGCSQICDNTPGSYHCSCKNGFVMLSNKKDCKDVDECSLKPSICGTAVCKNIPGDFECECPEGYRYNLKSKSCEDIDECSENMCAQLCVNYPGGYTCYCDGKKGFKLAQDQKSCE（配列番号１６）；および

Gla and EGF-like domains of human Pros1 without signal peptide and pro-domain:
ANSLLEETKQGNLERECIEELCNKEEAREVFENDPETDYFYPKYLVCLRSFQTGLFTAARQSTNAYPDLRSCVNAIPDQCSPLPCNEDGYMSCKDGKASFTCTCKPGWQGEKCEFDINECKDPSNINGGCSQICDNTPGSYHCSCKNGFVMLSNKKDCKDVDECSLKPSICGTAVCKNIPGDFECECPEGYRYNLKSKSCEDIDECSENMCAQQLCVNYPGGYTCYCDKDKK (sequence number 1)
is included.

In some non-limiting aspects of the invention, the fusion molecule comprises type I and type III IFN proteins or portions thereof. Type I IFN proteins for use in fusion molecules of the invention include IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-ε (epsilon), and IFN-ω (omega) or Some of which include, but are not limited to. Non-limiting exemplary mature type I IFN proteins are:

Human IFN-α2a:
and

Human IFN-β:
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN (SEQ ID NO: 19)
is.

Type III IFNs include IFN-λ1, IFN-λ2, IFN-λ3 and IFN-λ4 and portions thereof. Non-limiting exemplary type III IFN proteins are:

Human IFN-λ1:
PVPTSKPTPTGKGCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYVADGNLCLRTSTHPEST (SEQ ID NO:20); and

Human IFN-λ3:
VPVARLRGALPDARGCHIAQFKSLSPQELQAFKRAKDALEESLLLKDCKCRSRLFPRTWDLRQLQVRERPVALEAELALTLKVLEASADTDPALGDVLDQPLHTLHHILSQLRACIQPQPTAGPRTRGRLHHWLYRLQEAPKKESPGCLEASVTFNLFRLLTRDLNCVASGDLCV (SEQ ID NO: 21)
is.

In some aspects of the invention, the fusion molecule may further comprise a linker between the cytokine and the polypeptide that targets the fusion molecule to PS. Linkers for use in the present fusion molecules are preferably flexible and have a length ranging from 5-50 amino acids, or more preferably 10-30 amino acids. In some embodiments, the linker element is a glycine/serine linker, ie, a peptide linker consisting essentially of the amino acids glycine and serine. Amino acids threonine or alanine can also be used in the linker. It is clear to those skilled in the art that if a cytokine such as IFN already terminates with eg a Gly at the N-terminus of the fusion molecule, such a Gly may form the first Gly of the linker in the linker sequence. deaf. Similarly, if a cytokine such as IFN begins at the C-terminus with, for example, Pro, such a Pro residue may form the last Pro of the linker in the linker sequence. Examples of specific linker sequences are listed in Table 1. In a specific embodiment, the linker of the fusion molecule of the invention is set forth in SEQ ID NO:42.

Thus, in one non-limiting embodiment, the fusion molecule of the present invention is an N-terminal PS-linked domain with the signal peptide and pro-domain of mouse GAS6 fused to mouse IFN-β and mouse IFN-λ2 proteins. and an EGF-like oligomerization domain.

Gas6(Gla+EGF)-linker-IFN-β-linker-IFN-λ2 (Gas6(Gla+EGF)-IFN-β-IFN-λ2):
(SEQ ID NO:49).

In another non-limiting embodiment, the fusion molecule of the present invention comprises an N-terminal PS-linked domain with the signal peptide and pro-domain of human GAS6 and EGF fused to human IFN-β and human IFN-λ3 proteins. and a polypeptide comprising a similar oligomerization domain.

Gas6(Gla+EGF)-linker-IFN-β-linker-IFN-λ2 (Gas6(Gla+EGF)-IFN-β-IFN-λ2):
(SEQ ID NO:50).

Non-limiting embodiments of various fusion molecules of the invention are depicted in FIG. There are polypeptides, linkers and cytokines that target the fusion molecule to PS; polypeptides that target the fusion molecule to PS, linkers and a combination of two different cytokines; fusion molecules to PS. further comprising a domain that promotes oligomerization of the PS-binding domain upon binding to PS bound thereto, a linker and a cytokine; and targeting the fusion molecule to PS A polypeptide that binds to PS, further comprising a domain that promotes oligomerization of the PS-binding domain upon binding to PS bound thereto, a linker and a combination of two different cytokines. .

FIG. 2 depicts a model of cytokine receptor complexes and signaling pathways by the type III IFN (IFN-λ) and type I IFN (IFN-α/β) receptor systems exemplified herein. there is IFN-λ and type I IFNs use different heterodimeric receptor complexes. IFN-λ associates with native IFN-λR1 and IL-10R2, whereas IFN-αR1 and IFN-αR2 form the active type I IFN receptor complex. Engagement of IFN-α or IFN-λ receptors results in phosphorylation of the receptor-associated JAK kinases JAK1 and Tyk2, followed by phosphorylation of STAT1 and STAT2, which interact with the DNA-binding protein IRF9, resulting in the release of IFNs. It leads to the formation of a transcriptional complex called stimulatory gene factor 3 (ISGF3), which binds to the IFN-stimulated response element (ISRE) to control transcription of the IFN-stimulated gene (ISG).

FIG. 3 provides a schematic representation of non-limiting aspects of predicted binding and interactions of fusion molecules of the invention in PS-rich environments such as tumor microenvironments or sites of viral infection. As depicted in FIG. 3, in the PS-positive tumor microenvironment, the Gla domain of Gas6 directly binds to PS in apoptotic tumor cells or tumor vasculature and induces IFN-β and/or IFN-λ2. Immunostimulatory cytokines such as IFN-β and/or IFN-λ receptors on antigen presenting cells (APCs), tumor cells, endothelial cells and other tumor infiltrating cells will bind to their respective IFN-β and/or IFN-λ receptors.

Mechanisms of IFN-mediated anti-tumor activity include: i) suppressing its proliferation and promoting its apoptosis; ii) promoting the production of inflammatory cytokines and chemokines leading to increased recruitment of immune cells to tumors; and iii) antigen presentation by tumor cells achieved by upregulation of MHC class I and costimulatory molecules and changes in antigen processing, which results in an altered and diverse repertoire of tumor antigens presented by tumor cells. A direct effect on the tumor cells is included, increasing their size, which in turn leads to better recognition by T cells. IFNs also inhibit tumor angiogenesis by directly inhibiting endothelial cell proliferation and by stimulating the production of anti-angiogenic chemokines by tumor cells and tumor-infiltrating immune cells.

In addition, IFNs exert various immunostimulatory activities in immune cells, including: i) activation and expansion by professional antigen-presenting cells (APCs), leading to stimulation of type 1 helper T (Th1) cell responses; ii) stimulation of proliferation and differentiation of CD4+ and CD8+ T cells by direct action on these cells; iii) direct stimulation of NK cells and promotion of their anti-tumor activity.

Furthermore, PS-targeted cytokines may compete for PS binding by endogenous PS ligands such as Gas6 and Pros1, thus blocking the ability of Gas6 and Pros1 to induce immunosuppressive signals through TAM receptors. deaf. The fusion molecules of the invention will induce receptor clustering and result in stronger pStat1 signaling compared to IFN-β and IFN-λ2 alone. The fusion molecules of the invention therefore serve a dual function of targeted therapy and immunotherapy by binding to immunosuppressive PS molecules and inducing immunogenic signaling through cytokine receptors in the tumor microenvironment. Will.

The PS-targeting molecules of the present invention bind PS, but rather they engage immunosuppressive pathways through TAM receptors and activate IFN receptors to enhance host anti-tumor and anti-viral immunity. (Fig. 4).

A series of 12 murine Gas6-IFN fusion molecules containing either the Gla domain alone or both the Gla and EGF-like domains (Gla+EGF) of Gas6 were cloned and sequenced. Six mouse Gas6-IFN fusion molecules are depicted in FIG. Another variant contains a His-tagged protein, which not only facilitates its purification, but also allows the tag to be labeled for protein distribution imaging in vivo (Fig. 7). . All chimeric proteins were subsequently expressed in HEK293T cells and shown to be secreted into conditioned media, but were shown to be highly carboxylated, indicating that the cells is an essential post-translational modification required for fusion molecules that bind PS when cultured in the presence of vitamin K.

Figure 5 demonstrates the presence of several His-tagged Gas6-IFN fusion molecules in the conditioned medium of HEK293 cells transfected with the corresponding expression plasmids and their production and secretion from the cells (Figure 5(A)). ) as well as γ-carboxylation (FIG. 5(B)). Furthermore, dimer formation was observed to be strongly promoted in the presence of Gas6-derived EGF repeats. All Gas6-IFN proteins retain biological activity as shown by their ability to induce IFN signaling in reporter cell lines, and all bind PS in a γ-carboxylation-dependent manner. had the ability. Furthermore, the PS-binding domain of Gas6 (Gla-EGF-like domain), when fused with IFN, allows IFN to induce stronger signaling in the presence of apoptotic cells, suggesting that Gas6-IFN fusion molecules The strength of the IFN response evoked by is enhanced by increasing PS concentration, as intended by rationale and design (see Figures 6 and 8 and 19A to 19C). Furthermore, IFN activity is co-precipitated with apoptotic cells only when the Gas6-IFN fusion molecules are γ-carboxylated, so only γ-carboxylated Gas6-IFN fusion molecules bind apoptotic cells ( Figure 8).

In addition, mouse models of breast tumor growth in which mouse breast cancer E0771 cells were orthotopically implanted into the mammary fat pad were Gas6(Gla+EGF)-IFN-λ2 (see FIGS. 21A and 21B) and Gas6(Gla+EGF)-IFN-β showed that EO771 tumor cells, which constitutively express and secrete, exhibited growth retardation when injected into the mammary fat pad of syngeneic immune competent C57BL/6 mice. The Gas6(Gla+EGF)-IFN-λ2 fusion molecule showed a significant reduction in tumor volume compared to controls, also referred to herein as mock. A Gas6(Gla+EGF)-IFN-λ2 fusion molecule secreted from EO771 cells has also been shown in vitro to have IFN-λ2 activity in IFN-λ reporter cells.

Furthermore, a mouse model of breast tumor growth also showed that the Gas6(Gla+EGF)-IFN-β-IFN-λ2 fusion molecule has anti-cancer activity comparable to that of the IFN-β-IFN-λ2 fusion molecule. (See Figures 16 and 17). In this model, EO771 mammary tumor cells, which constitutively express and secrete Gas6(Gla+EGF)-IFN-β-IFN-λ2 or IFN-β-IFN-λ2 molecules, were isolated from syngeneic immune competent C57BL/6 mouse mammary glands. It showed growth retardation when injected into the fat pad. Tumor cells expressing either Gas6(Gla+EGF)-IFN-β-IFN-λ2 or IFN-β-IFN-λ2 fusion molecules significantly reduced tumor volume compared to mock-transfected tumor cells , and 4 out of 8 mice remained tumor-free in each group (FIGS. 16 and 17).

Furthermore, EO771 cells expressing the Gas6-IFN-β-IFN-λ2 fusion molecule were more potent than a 50:50 mixture of EO771 cells that constitutively secrete the Gas6-IFN-β and Gas6-IFN-λ2 proteins. , grow very slowly in vivo and the fusion Gas6-IFN-β-IFN-λ2 molecule was shown to have higher anti-tumor potency than the combination of PS-targeted type I and III IFNs. was done.

Are the antiviral activities of the PS-targeted IFN fusion molecules of the invention comparable to the native protein (FIG. 20A; Gas6(Gla)-IFN-λ2 and Gas6(Gla+EGF)-IFN-λ2 against IFN-λ2)? 10 and 11 (Gas6(Gla+EGF)-IFN-β-IFN-λ2 to IFN-β-IFN-λ2) to be more potent than the IFN fusion molecule alone. was either In addition, the ability of the fusion molecules of the invention to induce IFN receptor responses induced by the expression of the immunostimulatory proteins calreticulin and MHC class I proteins and the immunomodulatory PD-L1 protein, respectively. 12, 20C, 20B and 13.

The fusion molecules of the invention can be produced by conventional recombinant expression methodologies using known expression systems, including E. coli, yeast, baculovirus, insect, plant or mammalian protein expression systems. is not limited to Fusion molecules can be recovered and purified from recombinant cell culture by any effective method. For example, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphate cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. See, eg, Lin, et al. (1986) Meth. Enzymol. 119: 183-192. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification. Additionally, methods that can be used to produce and isolate fusion molecules of the invention are disclosed in US Pat. No. 6,433,145.

Additionally, the fusion molecules of the invention can be chemically synthesized using any available technique (see, e.g., Creighton (1983) Proteins: Structures and Molecular Principles, W.H. Freeman & Co., NY; Hunkapiller, et al.). al. (1984) Nature 310:105-111). For example, fusion molecules or fragments of fusion molecules can be synthesized using a peptide synthesizer.

The invention also includes, for example, gamma-carboxylation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, binding to antibody molecules or other cellular ligands, etc. It also includes fusion molecules that are modified during or after translation by . specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; Any number of chemical modifications can be made by the technique of

Additional post-translational modifications encompassed by the present invention include, for example, gamma-carboxylation, N-linked or O-linked glycans, N-terminal or C-terminal processing, chemical moieties to the amino acid backbone. , chemical modification of N-linked or O-linked carbohydrate chains, and addition or removal of N-terminal methionine residues as a result of prokaryotic host cell expression. Fusion molecules may also be modified with detectable labels such as enzymatic, fluorescent, isotopic or affinity labels that allow detection and isolation of proteins.

Also provided by the invention are chemically modified derivatives of the fusion molecules of the invention, which have additional advantages such as increased solubility, stability and circulation time of the polypeptide or decreased immunogenicity. (see U.S. Pat. No. 4,179,337). Chemical moieties for derivatization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol. Polypeptides may be modified at random positions within the molecule or at predetermined positions within the molecule and may contain one, two, three or more chemical moieties attached. .

The polymer can be of any molecular weight and can be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa for ease of handling and manufacturing (the term "about" is used in the preparation of polyethylene glycol to indicate that a molecule is heavier than the stated molecular weight). , indicating that some molecules may be lighter). The desired therapeutic profile (e.g., desired duration of sustained release, effect, if any, on biological activity, ease of handling, degree or lack of antigenicity, and polyethylene glycol for therapeutic proteins or analogs). Other sizes may be used for other known effects). For example, polyethylene glycol has a , 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16 , 500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000 , 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa. good.

As noted above, polyethylene glycol may have a branched structure. Branched polyethylene glycols are disclosed, for example, in U.S. Pat. No. 5,643,575; Morpurgo, et al. (1996) Appl. Biochem. Biotechnol. 56:59-72; Vorobjev, et al. (1999) Nucleosides Nucleotides 18: 2745-2750; and Caliceti, et al. (1999) Bioconjug, Chem. 10:638-646.

Polyethylene glycol molecules (or other chemical moieties) should be attached to the fusion molecule in view of their effect on the functional or antigenic domains of the protein. There are numerous attachment methods available to those skilled in the art, for example EP 0 401 384, which teaches the coupling of PEG to G-CSF, and PEGylation of GM-CSF with tresyl chloride. See Malik, et al. (1992) Exp. Hematol. 20:1028-1035, described. For example, polyethylene glycol can be covalently attached to amino acid residues through reactive groups such as free amino or carboxyl groups. A reactive group is one to which an activated polyethylene glycol molecule can be attached. Amino acid residues with free amino groups may include lysine residues and N-terminal amino acid residues; those with free carboxyl groups include aspartic acid residues, glutamic acid residues and C-terminal residues. may be included. Sulfhydryl groups can also be used as reactive groups for attaching polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at the amino group, such as attachment at the N-terminus or lysine group.

As noted above, polyethylene glycol may be attached to proteins via linkages to any of a number of amino acid residues. For example, polyethylene glycol can be attached to proteins via covalent linkages to lysine, histidine, aspartate, glutamate, or cysteine residues. Polyethylene glycol is attached to a specific amino acid (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) within a protein, or to two or more amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine, and combinations thereof), one or more reaction chemistries may be used.

In some cases, proteins chemically modified at the N-terminus are particularly desired. Using polyethylene glycol as described, from a variety of polyethylene glycol molecules (due to molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mixture, the type of pegylation reaction to be performed, and a method to obtain the selected N-terminal PEGylated protein. A method of obtaining an N-terminal pegylated preparation (i.e., optionally separating this moiety from other monopegylated moieties) may be by purification of the N-terminal pegylated material from a population of pegylated protein molecules. . Selective proteins that have been chemically modified with N-terminal modifications are reductive alkylations that exploit the differential reactivity of different species of primary amino groups (lysine to N-terminus) available for derivatization in a given protein. can be achieved by Under appropriate reaction conditions, substantial selective derivatization of proteins at the N-terminus is achieved with carbonyl group containing polymers.

As indicated above, pegylation of the fusion molecules of the invention can be accomplished by a number of means. For example, polyethylene glycol can be attached to proteins either directly or via an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al. (1992) Crit. Rev. Thera. Drug Carrier Sys. 9:249-304; Francis, et al. (1998) Intern. 68:1-18; U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466. ing.

The number of polyethylene glycol moieties (ie, degree of substitution) attached to the fusion molecules of the invention may also vary. For example, the pegylated proteins of the invention are conjugated to, on average, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules. may Similarly, the average degree of substitution per protein molecule is 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10- Within ranges such as 12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties. Methods for determining the degree of substitution are discussed, for example, in Delgado, et al. (1992) Crit. Rev. Thera. Drug Carrier Sys. 9:249-304.

The fusion molecules of the invention can be used to treat a variety of cancers, viral diseases and other indications, particularly those in which the lesion site is rich in PS.

Therefore, the present invention also provides for targeting cytokines or portions thereof to the lesion site of interest, inhibiting immunosuppression resulting from PS recognition by endogenous PS ligands and receptors at the lesion site of interest, activating one or more cytokine-specific biological activities, minimizing systemic effects of cytokines in the subject, and/or administering to the subject an effective amount of the fusion molecule or pharmaceutical composition comprising the fusion molecule. Also provided are pharmaceutical compositions and methods for treating a disease, disorder or condition responsive to cytokine treatment in a subject via. In one non-limiting embodiment, the disease, disorder or condition targeted and/or treated by the present invention is cancer, infection or an inflammatory condition or disorder.

For purposes of the present invention, a "subject" is a mammalian animal such as a human, non-human primate (e.g., baboon, orangutan, monkey), mouse, pig, cow, goat, cat, rabbit, rat, guinea pig, hamster. , horses, monkeys, sheep; or non-mammalian animals, including non-mammalian vertebrates such as, for example, birds (eg, chickens or ducks) or fish, and non-mammalian invertebrates.

According to the methods of the present invention, "effective amount" means a dose or amount of a fusion molecule or pharmaceutical composition comprising a fusion molecule sufficient to produce a desired result. A desired result can include an objective or subjective improvement in a subject receiving the dose or amount. In particular, an effective amount is an amount that suppresses, alleviates, reduces or eliminates one or more signs or symptoms associated with a disease or condition. Treatment may include treatment of existing symptoms or prophylaxis of anticipated infections, including but not limited to general recurrence of infections such as influenza, and emergency prophylaxis as circumstances call for, such as bioweapon attacks. is included.

In some non-limiting embodiments, the methods of the invention are used to treat chronic hepatitis C infection, chronic hepatitis B infection, herpes virus, papilloma virus, influenza A virus, influenza B virus, respiratory syncytial virus, Chronic and acute viral infections such as, but not limited to rhinovirus, coronavirus, rotavirus, norovirus, enterovirus, Zika virus, Ebola virus, dengue virus, chikungunya virus, hantavirus and AIDS/HIV; breast, bone, liver cancer, including but not limited to solid tumors, including sarcomas, carcinomas, and lymphomas of the kidney, lung, neck and throat, skin, colon, prostate, bladder and pancreas; and Crohn's disease, multiple sclerosis and It is used in the treatment of inflammatory and/or autoimmune conditions or diseases such as, but not limited to, arthritis, asthma, psoriasis, dermatitis, autoimmune pneumonia or gastroenteritis, condyloma acuminata. In certain non-limiting embodiments, the fusion molecules and methods of the invention are used in the treatment of viral infections or cancer.

Any effective amount of a fusion molecule of the invention may be administered to a subject in need thereof, eg, a subject having or at risk of acquiring a disease or condition. As a general proposition, the total parenterally administered pharmaceutically effective amount per dose would be about 1 μg/kg/day to 10 mg/kg/day of the patient's body weight, but as noted above: This would be subject to therapeutic discretion. More preferably, the dose is at least 0.01 mg/kg/day, and most preferably between about 0.01 and 1 mg/kg/day for humans. When given continuously, the composition will typically be from about 1 μg/kg/hour to about 50 μg/kg, either by 1-4 injections per day or by continuous subcutaneous infusion, eg, using a minipump. /hour dosing rate. Intravenous bag solutions can also be used. The duration of treatment required to observe changes and the interval between subsequent treatments for response to occur may vary depending on the desired effect.

For therapeutic purposes, the fusion molecules of the invention are preferably provided as pharmaceutical compositions containing the fusion molecule in admixture with a pharmaceutically acceptable carrier. The term "pharmaceutical composition" means a composition suitable for pharmaceutical use in subjects, including animals or humans. Pharmaceutical compositions generally comprise an effective amount of an active agent and a pharmaceutically acceptable carrier such as, for example, a non-toxic solid, semisolid or liquid filler, carrier, diluent, encapsulating material or Including auxiliaries for all kinds of formulations.

Pharmaceutical compositions containing the fusion molecules of the invention can be administered, for example, orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally (powder, ointment, infusion). It may be administered by any effective route, including topically, buccally or as an oral or nasal spray (or via a transdermal patch).

The term "parenteral" as used herein includes any effective parenteral mode of administration, including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion modes. Say.

The compositions may also suitably be administered by sustained release systems. Suitable examples of sustained release compositions include semipermeable polymer matrices in the form of shaped articles, eg films, or microcapsules. Sustained-release matrices include polylactic acid (U.S. Pat. No. 3,773,919; European Patent Application Publication No. 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al. Biopolymers 1983 22:547-556), poly(2-hydroxyethyl methacrylate) (Langer et al. J. Biomed. Mater. Res. 1981 15:167-277; Langer Chem. Tech. 1982 12:98-105). ), ethylene vinyl acetate or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).

Sustained-release compositions also include liposome-encapsulated polypeptides. Liposomes containing the polypeptides of the invention are prepared by methods known in the art German Patent Application No. 3,218,121; Epstein, et al. Proc. Natl. Acad. Sci. USA 1985 USA 1980 77:4030-4034; EP-A-52,322; EP-A-36,676. European Patent Application Publication No. 88,046; European Patent Application Publication No. 143,949; European Patent Application Publication No. 142,641; 4,485,045; U.S. Pat. No. 4,544,545; and European Patent Application Publication No. 102,324. Ordinarily, liposomes are of the small (about 200-800 angstroms) unilamellar type and have a lipid content greater than about 30 mole percent cholesterol, the selected proportion being adjusted for effective polypeptide therapy. .

When used as an immuno-oncological agent (ICI), the fusion molecules of the invention may be used alone or in combination with other ICIs. In one non-limiting embodiment, the fusion molecules of the invention may be used in combination with anti-PD-1 therapy. This combination is particularly attractive because type I and type III IFNs induce upregulation of PD-L1, an effect that may reduce the anti-tumor efficacy of the fusion molecule.

When used as antiviral agents, the fusion molecules of the invention may be administered alone or in combination with other known antiviral, immunomodulatory agents such as IL-2, KDI, ribavirin and temozolomide. and may be administered in combination with anti-proliferative therapies.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Such containers may be accompanied by a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, and such notice shall be issued by the agency for administration to humans. It reflects an authorization to make, use or sell by Additionally, the fusion molecules of the invention may be used in conjunction with other therapeutic compounds.
The following non-limiting examples are provided to further illustrate the invention.

Examples Example 1: Synthetic Methods of Fusion Molecules Intact unmodified GAS6, IFN-λ2, IFN-β, and a fusion IFN molecule, IFN-β-IFN, were used to generate fusion molecules for biological evaluation. -λ2 (control) and six GAS6-IFN fusion molecules Gas6(Gla)-IFN-λ2, Gas6(Gla)-IFN-β, Gas6(Gla)-IFN-β-IFN-λ2, Gas6( Mammalian plasmids expressing Gla+EGF)-IFN-λ2, Gas6(Gla+EGF)-IFN-β and Gas6(Gla+EGF)-IFN-β-IFN-λ2 were transiently transfected into HEK293T cells. Plasmids expressing His-tagged and phosphorylatable versions of the fusion molecule were also constructed and transfected into HEK293 cells. Conditioned media containing secreted intact or fusion proteins were harvested 48 hours after transfection.

Example 2 Immunoblotting Methods Conditioned media containing various intact Gas6 and IFN molecules and Gas6(Gla), or Gas6(Gla+EGF) IFN fusion molecules were separated by SDS-PAGE, transferred to membranes, and gamma carboxyl γ-Carboxylation was assessed by immunoblotting with antibodies specific for glycosylation and His-tag.

Example 3 Evaluation of γ-Carboxylation and Cytokine Activity The activity of Gas6(Gla), or Gas6(Gla+EGF) IFN fusion proteins was assessed by treating IFN-λR-γR1 reporter cell lines. Reporter cells were treated for 30 minutes with recombinant IFN-λ2, which was used as a control, or HEK293T cell supernatant containing Gas6(Gla) and Gas6(Gla+EGF) IFN fusion molecules with or without apoptotic cells. Cell lysates were prepared and Stat1 phosphorylation was measured by immunoblotting with antibodies specific for tyrosine-phosphorylated Stat1 (pStat1) as an output of IFN-λ receptor activation. A pStat1 immunoblot showed a phosphatidylserine binding-dependent increase in IFN-λ receptor activation by the fusion molecule. Furthermore, binding of apoptotic cells to γ-carboxylated Gas6-IFN fusion molecules was demonstrated by co-precipitation of IFN activity with apoptotic cells.

Example 4: Antiviral Activity Equal numbers of human retinal pigment epithelial ARPE19 cells or mouse intestinal epithelial cells (mIEC) were seeded in all wells of a 96-well microtiter plate in DMEM medium containing 10% FCS and 300 ng/ml-0 by recombinant IFN-λ2 at various concentrations ranging from .002 ng/ml, or by Gas6(Gla)-IFN-λ2 and Gas6(Gla+EGF)-IFN-λ2 fusion molecules, or IFN-β-IFN-λ2 and Gas6( Gla+EGF)-IFN-β-IFN-λ2 were treated with 3-fold serial dilutions of HEK293T cell supernatants, respectively. Twenty-four hours after pretreatment, the cells were loaded with vesicular stomatitis virus (VSV) added to the wells at a concentration of 0.1 pfu/cell and the cells were incubated for an additional 24 hours to determine the antiviral activity of the fusion molecule. was analyzed. Cell viability was measured using the MTT assay according to the manufacturer's protocol (Millipore/Sigma).

ARPE-19 cells or mIEC were also seeded in 6-well plates in DMEM medium containing 10% FCS, either untreated or with recombinant IFN-λ2 (100 ng/ml) or Gas6(Gla)-IFN- λ2, Gas6(Gla+EGF)-IFN-λ2, IFN-β-IFN-λ2 or Gas6(Gla+EGF)-IFN-β-IFN-λ2 fusion molecules were treated with 1/10 dilutions of HEK293T cell supernatants. and left for 72 hours. Cells were then harvested and cell surface level MHC class I antigen expression, calreticulin expression or PD-L1 expression was measured by flow cytometry.

Example 5: Anti-tumor activity 10 ⁵ EO771 mock cells (EO771 cells transfected with empty vector) in immunocompetent syngeneic 6-8 week old C57BL/6 mice (Jackson Laboratory) IFN-β-IFN - λ2 Gas6(Gla+EGF)-IFN-β, Gas6(Gla+EGF)-IFN-λ2 or Gas6(Gla+EGF)-IFN-β-IFN-λ2 fusion molecule-secreting cells (transfected with vectors expressing various fusion molecules) EO771 cells) were injected into the mammary fat pad. Mice were checked for tumor growth by palpation of the injection site every 1-2 days, tumor volume (V) was measured using clippers, tumor length (L) and width (W) were measured, It was then calculated by applying the formula V = (LxWxW)/2.

Example 6 Evaluation of the Ability of Fusion Molecules to Recruit to and Localize in the Tumor Microenvironment To determine whether the fusion molecule could be specifically delivered to the tumor site, an evaluation was performed to determine whether the fusion molecule could be delivered specifically to the tumor site. These studies include the Mx2 luciferase reporter transgenics, whose luciferase expression is controlled by the IFN-inducible Mx2 promoter, described by Pulverer, JE et al. (Journal of Virology 2010 84:8626-8638). TG) mice are used. These reporter mice express luciferase in a tissue-specific manner when injected intravenously with either type I or type III IFNs: type I IFNs induce luciferase expression primarily in the liver, Type III IFNs cause luciferase expression in the gastrointestinal tract (McElrath, C. et al. Cytokine 2016 87:141-141).

His-tagged proteins are produced in HEK293 cells and purified to homogeneity. Intact and fusion IFN proteins are initially injected into reporter mice at various concentrations, and luciferase location and duration are monitored in surviving animals using, for example, the Xenogen IVIS 200 Imaging System. Female mice were then injected with E0771 cells into the mammary fat pad and after tumor establishment, mice were injected with intact or Gas6-fused IFN molecules and luciferase expression was assessed in surviving animals.

Under physiological conditions, few non-depleted apoptotic cells and PS-positive stress cells are observed, even in tissues with high cell turnover rates such as thymus and spleen. This is because cells undergoing apoptosis as part of normal homeostasis are efferocytosed so efficiently and strongly that PS is undetectable in normal tissues. Therefore, PS targeting of the fusion molecules of the invention determined by this study localizes the designed fusion molecules to sites where PS is upregulated as part of stress responses and cancer, viral infection or inflammation. indicates targeted delivery.

Example 7 Evaluation of Ability of Fusion Molecules to Recruit and Localize in Tumor Microenvironment and Sites of Viral Infection and injected by tail vein or SQ injection into tumor-bearing mice and mice infected with either respiratory influenza A virus or gastrointestinal rotavirus, and the in vivo distribution of the labeled protein was determined by X Monitoring was performed by line imaging and measuring radioactivity distribution in various anatomical tissues.

Example 8: Anti-Tumor Efficacy of Fusion Molecules in Two Mouse Models The tumor microenvironment-altering properties of the fusion molecules of the invention were tested in two independent, genetically receptive, orthotopic breast cancer growth studies. Compare in a sex transplant model. These models include the 4T1 cell model (for the BALB/c mouse strain) and the E0771 cell model (for the C57BL/6 mouse strain). Both models reflect a malignant, triple negative tumor breast cancer model that recapitulates aspects of human breast cancer, including low immunogenic potential and spontaneous metastasis to the lung. Furthermore, both 4T1 and E0771 cells are thought to express all three TAMs, making these cancer models suitable for studying tumors that express PS receptors. 4T1 and E0771 cells were generated that constitutively express various intact or Gas6-fused IFN molecules. Cell populations are selected that express comparable levels of IFN molecules.

Growth kinetics of modified cells are first compared in vitro. For animal studies, 8-week-old syngeneic wild-type C57BL/6 (E0771) or BALB/c (4T1) female virgin mice were injected with 10 ⁵ mouse breast cancer cell lines (resuspended in 50% Matrigel). , injected in the center of the right 4th inguinal mammary fat pad (n=12 mice/group). Primary tumor volumes are assessed and recorded every other day. When primary tumors reach 1 cm ³ , mice are sacrificed and lung metastases are quantified. Lungs, bones, brains and other major organs were weighed, half snap-frozen and half fixed for further biochemical and histological analysis, proliferation (Ki67), apoptosis (Tunnel), microvascular (CD31) and PASR staining. Potential spontaneous metastases will be dissected and biochemically analyzed using laser micro-capture techniques as appropriate.

Example 9: Evaluation of the effect of fusion molecules on immune cell frequency in TME Examining subsets of immune cells in TME and how these are altered by the fusion molecules of the invention may alter immune responses. provide mechanistic insight into their role in It is expected that the fusion molecule will reverse inhibitory signals that affect the host's anti-tumor response, reprogramming the TME into a more immunocompetitive environment. To address these questions experimentally, a combination of Nanostring and IHC-based methods are used to probe the cellular frequencies of PMNs, DCs, MPhs, NKs and T cells in the TME and at the tumor border.

Therefore, upon removal of the primary tumor, an aliquot was used to examine the tumor borders by IHC, followed by enzymatic digestion to isolate the tumor and tumor-infiltrating cells to isolate F4/80 MPh, GR1+ neutrophils, CD11+ DC. and T cells, myofibroblasts and endothelial cells (PECAM+ cells). Leukocyte (DC, MPh, NK and T cell) infiltration and DC maturation status at the tumor site were also determined by immunostaining cells followed by CD86 (Alexa 350 labeling) for DC, F4/80 (Alexa 405 labeling) for MPh. labeling) and by FACS analysis (BD LSR II) using specific markers such as CD4+ (PE-Cy7 labeling) and CD8+ (Alexa 649 labeling) for T cells. In addition, tumor-associated cytokines and chemokines are quantified by MSD-cytokine array (Meso Scale Diagnostics, Rockville, Md.).

Example 10 Evaluation of Therapeutic Efficacy of Fusion Molecules in Tumor Growth Animal Models Fusion molecules demonstrating the most potent anti-tumor efficacy as anti-cancer therapeutics are further tested in the models described above. For these experiments, fusion molecules are produced and purified endotoxin-free using His-tag purification technology in quantities sufficient for animal testing. For these experiments, original 4T1 and E0771 tumors are established and grown to a volume of approximately 0.3 cm ³ and animals are injected intravenously daily with 1 μg of the selected purified fusion molecule. Lower doses and frequencies of recombinant protein administration are also tested if effective tumor suppression is achieved.

Example 11 Evaluation of Therapeutic Efficacy of Fusion Molecules in Animal Models of Viral Infection The antiviral efficacy of PS-targeted IFN fusion molecules is tested using a mouse model of influenza A infection. Efficacy is compared to intact IFN. 8-24 hours prior to infection of mice with 1 LD ₅₀ of influenza A virus PR8, WSN, Udorn strain or other strains, prophylactically at various doses (0.1 per approximately 20 mg adult 8-week-old mouse) , 0.3, 1, 3, 10 μg; PBS is used as control mock treatment) mice are injected SQ or intranasally (IN). Survival and weight loss are monitored daily.

In addition, virus titers and lung tissue lesions are assessed at 3, 6 and 9 days post-infection in a separate experiment. Histological lesions are used to assess lesions. IHC staining of viral antigens is used to determine if the pattern of viral transmission changes with treatment. Optimal IFN treatment to increase post-infection survival will also be evaluated. In this experiment, the efficacy of treatment following infection with influenza A virus (1LD50 PR8, WSN, Udorn strain or other strains) is tested at multiple dosing regimens. As above, mice were treated with SQ or intranasally (0.1, 0.3, 1, 3, 10 μg per adult approximately 20 mg 8-week-old mouse; PBS as a control mock treatment) at various doses. IN) Treat with an injected IFN fusion molecule, a single IFN, or a combination thereof. Infected mice are treated according to the following schedule: 1, 3, 5; 1-4; 2, 4, 6; 2-5 days. Mice are analyzed as above and scored for antiviral protection as well as disease progression.

Example 12 Evaluation of the Ability of Fusion Molecules to Inhibit Signaling of Intact TAM Ligand Through TAM Receptors Treat with intact γ-carboxylated Gas6 and Pros1 in the presence or absence of Gas6-IFN fusion molecules given in excess. The ability of fusion molecules to block TAM receptor activation by endogenous intact ligands is assessed by measuring the reduction in Stat1 activation (pStat1).

Claims (11)

a fusion molecule,
an interferon or an immunostimulatory portion thereof;
A polypeptide containing the N-terminal γ-carboxylated phosphatidylserine (PS)-binding Gla domain of growth arrest specific gene 6 (GAS6), which targets the fusion molecule to cell surface PS, and interferon or its said polypeptide actively altering the immune balance from PS-induced immunosuppression to immune stimulation in an expressed PS concentration-dependent manner by increasing and maintaining interferon receptor activation by the immunostimulatory moiety and the fusion molecule. 2. The fusion molecule of Claim 1 , wherein the polypeptide further comprises a domain that promotes oligomerization of the PS binding domain upon binding PS. 2. The fusion molecule of claim 1 , further comprising an epidermal growth factor (EGF)-like domain. 4. Any one of claims 1-3, wherein the interferon is selected from interferon-alpha, interferon-beta, interferon-lambda1, interferon-lambda2, interferon-lambda3, or combinations or parts thereof. fusion molecule. 5. The fusion molecule of any one of claims 1-4 , further comprising a linker between the interferon or immunostimulatory portion thereof and the polypeptide that targets the fusion molecule to PS. A pharmaceutical composition comprising the fusion molecule of any one of claims 1-5 and a pharmaceutically acceptable carrier. 7. The pharmaceutical composition of Claim 6 for targeting interferon or an immunostimulatory portion thereof to a lesion site in a subject. 8. The pharmaceutical composition according to claim 7 , wherein the lesion site is a PS-rich area and/or contains cancer, infection or inflammation. Immunosuppression resulting from PS recognition by endogenous PS ligands and receptors is inhibited at the lesion site of the subject; and/or one or more interferon -specific biological activities are activated at the lesion site and/or
systemic effects of the interferon or immunostimulatory portion thereof are minimized;
9. Pharmaceutical composition according to claim 7 or 8 . A pharmaceutical composition according to any one of claims 6 to 9 for treating diseases, disorders or conditions responsive to interferon treatment. 11. The pharmaceutical composition of claim 10, wherein the disease, disorder or condition responsive to interferon treatment is cancer, infection or an inflammatory condition or disease.

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